Evidence for a Causal Role for Escherichia coli Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation.

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation. IMPORTANCE Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria. Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients. However, whether this bloom contributes to disease or is just a response to IBD-associated physiological changes is unknown. Although assigning causality is challenging, appropriate animal models can test the hypothesis that AIEC strains have an enhanced ability to cause colitis in comparison to other gut commensal E. coli strains and to identify bacterial traits contributing to virulence. We observed that AIEC strains are generally more pathogenic than commensal E. coli and that bacterial intracellular survival/replication phenotypes contributed to disease. We also found that E. coli strains lacking primary virulence traits can prevent inflammation. Our findings provide critical information on E. coli pathogenicity that may inform development of IBD diagnostic tools and therapies.

Association of ISVsa3 with Multidrug Resistance in Salmonella enterica Isolates from Cattle (Bos taurus).

Salmonella enterica is, globally, an important cause of human illness with beef being a significant attributable source. In the human patient, systemic Salmonella infection requires antibiotic therapy, and when strains are multidrug resistant (MDR), no effective treatment may be available. MDR in bacteria is often associated with the presence of mobile genetic elements (MGE) that mediate horizontal spread of antimicrobial resistance (AMR) genes. In this study, we sought to determine the potential relationship of MDR in bovine Salmonella isolates with MGE. The present study involved 111 bovine Salmonella isolates obtained collectively from specimens derived from healthy cattle or their environments at Midwestern U.S. feedyards (2000-2001, n = 19), or specimens from sick cattle submitted to the Nebraska Veterinary Diagnostic Center (2010-2020, n = 92). Phenotypically, 33/111 isolates (29.7%) were MDR (resistant to ≥3 drug classes). Based on whole-genome sequencing (WGS; n = 41) and PCR (n = 111), a MDR phenotype was strongly associated (OR = 186; p < 0.0001) with carriage of ISVsa3, an IS91-like Family transposase. In all 41 isolates analyzed by WGS ((31 MDR and 10 non-MDR (resistant to 0-2 antibiotic classes)), MDR genes were associated with carriage of ISVsa3, most often on an IncC type plasmid carrying blaCMY-2. The typical arrangement was floR, tet(A), aph(6)-Id, aph(3″)-Ib, and sul2 flanked by ISVsa3. These results suggest that AMR genes in MDR S. enterica isolates of cattle are frequently associated with ISVsa3 and carried on IncC plasmids. Further research is needed to better understand the role of ISVsa3 in dissemination of MDR Salmonella strains.

Effect of potassium tellurite concentration in a chromogenic agar medium on isolation of tellurite-resistant "Top Seven" Shiga toxin-producing Escherichia coli from ground beef.

The effect of potassium tellurite concentration in a chromogenic agar medium on the detection of tellurite-resistant "top seven" Shiga toxin-producing Escherichia coli (STEC) in beef was evaluated. Samples of ground beef were inoculated with tellurite-resistant STEC O26, O45, O103, O111, O121, O145, or O157 strains at geometric mean (±standard error of the mean) levels of 0, 49 (±1), 490 (±1), or 4900 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against STEC serogroups; pool I: O26, O45, and O121; pool II: O103, O111, and O145; and pool III: O157. After immunomagnetic separation, 50 µL of washed bead suspensions in buffered peptone water was spiral plated onto a modified Possé medium containing 0.5, 1.0, or 1.5 mg/L potassium tellurite, and incubated at 37°C for 18 h. Up to four isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, across all inoculum levels and strains, modified Possé media containing 0.5, 1.0, or 1.5 mg/L potassium tellurite each had a positive predictive value of 100%, and medium containing 0.5 mg/L potassium tellurite had numerically the highest sensitivity (100%) and negative predictive value (100%), which was significantly different from 1.5 mg/L (92.9% and 40.0%, respectively; P < 0.05). Similarly, there was an inverse relationship between potassium tellurite concentration and analytical specificity (number of colonies tested that were STEC-positive): 0.5 (1463 of 1482; 98.7%), 1.0 (1356 of 1411; 96.1%), and 1.5 mg/L (1187 of 1278; 92.9%; P < 0.05). These results suggest that 0.5 mg/L gives better performance than 1.0 or 1.5 mg/L of potassium tellurite in Possé medium for isolation of tellurite-resistant "top seven" STEC from ground beef.

Isolation and characterization of Escherichia albertii in poultry at the pre-harvest level.

Escherichia albertii, often misidentified as Escherichia coli, has become an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. Here, we performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. Although no E. albertii was identified in a total of 1,022 diverse E. coli strains isolated from pets and food animals in a retrospective screening, in a pilot study, E. albertii was successfully isolated from a broiler farm (6 out of 20 chickens). The chicken E. albertii isolates showed clonal relationship as indicated by both pulsed-field gel electrophoresis (PFGE) and whole-genome sequence analysis. The isolated chicken E. albertii displayed multidrug resistance; all the resistance determinants including the extended-spectrum beta-lactamase gene, carried by plasmids, could be conjugatively transferred to E. coli, which was further confirmed by S1-PFGE and Southern hybridization. Whole-genome sequence-based phylogenetic analysis showed the chicken E. albertii strains were phylogenetically close to those of human origins. Challenge experiment demonstrated that the E. albertii strains isolated from human and wild bird could successfully colonize in the chicken intestine. Together, this study, for the first time, reported the isolation of E. albertii in poultry at the pre-hrvest level. The findings from multi-tier characterization of the chicken E. albertii strains indicated the importance of chickens as a reservoir for E. albertii. A large scale of E. albertii survey in poultry production at the pre-harvest level is highly warranted in the future.

Recovery Rate of Cells of the Seven Regulated Serogroups of Shiga Toxin-Producing Escherichia coli from Raw Veal Cutlets, Ground Veal, and Ground Beef from Retail Stores in the Mid-Atlantic Region of the United States.

ABSTRACT: A total of 482 veal cutlet, 555 ground veal, and 540 ground beef samples were purchased from retail establishments in the mid-Atlantic region of the United States over a noncontiguous 2-year period between 2014 and 2017. Samples (325 g each) were individually enriched and screened via real-time PCR for all seven regulated serogroups of Shiga toxin-producing Escherichia coli (STEC). Presumptive STEC-positive samples were subjected to serogroup-specific immunomagnetic separation and plated onto selective media. Up to five isolates typical for STEC from each sample were analyzed via multiplex PCR for both the virulence genes (i.e., eae, stx1 and/or stx2, and ehxA) and serogroup-specific gene(s) for the seven regulated STEC serogroups. The recovery rates of non-O157 STEC from veal cutlets (3.94%, 19 of 482 samples) and ground veal (7.03%, 39 of 555 samples) were significantly higher (P < 0.05) than that from ground beef (0.93%, 5 of 540 samples). In contrast, only a single isolate of STEC O157:H7 was recovered; this isolate originated from 1 (0.18%) of 555 samples of ground veal. Recovery rates for STEC were not associated with state, season, packaging type, or store type (P > 0.05) but were associated with brand and fat content (P < 0.05). Pulsed-field subtyping of the 270 viable and confirmed STEC isolates from the 64 total samples testing positive revealed 78 pulsotypes (50 to 80% similarity) belonging to 39 pulsogroups, with ≥90% similarity among pulsotypes within pulsogroups. Multiple isolates from 43 (67.7%) of 64 samples testing positive had an indistinguishable pulsotype. STEC serotypes O26 and O103 were the most prevalent serogroups in beef and veal, respectively. These findings support related findings from regulatory sampling studies over the past decade and confirm that recovery rates for the regulated STEC serogroups are higher for raw veal than for raw beef samples, as was observed in the present study of meat purchased at food retailers in the mid-Atlantic region of the United States.

Special Issue: Shiga Toxin-Producing Escherichia coli.

Globally, Shiga toxin-producing Escherichia coli (STEC) is an important cause of diarrheal disease, most notably hemorrhagic colitis, and post-diarrheal sequela, such as hemolytic-uremic syndrome (HUS) [...].

Correction: Moxley, R.A., et al. Intimate Attachment of Escherichia coli O157:H7 to Urinary Bladder Epithelium in the Gnotobiotic Piglet Model. Microorganisms 2020, 8, 263.

The authors wish to make the following corrections to this paper [...].

Performance of Chromogenic Agar Media for Isolation of Shiga Toxin-Producing Escherichia coli from Ground Beef.

ABSTRACT: The performance of three chromogenic agar media for detection of the "top seven" Shiga toxin-producing Escherichia coli (STEC) in beef was compared. Samples of retail ground beef were inoculated with STEC O26, O45, O103, O111, O121, O145, or O157 at geometric mean (±standard error of the mean) levels of 0, 48 (±1), 420 (±1), 4,100 (±1), or 45,000 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against the seven STEC serogroups: pool I, O26, O45, and O121; pool II, O103, O111, and O145; and pool III, O157. After immunomagnetic separation, 50 µL of washed bead suspensions in buffered peptone water were spiral plated onto modified Rainbow Agar O157 (mRBA), CHROMagar STEC (CS), or modified Possé differential medium (mPossé2) and incubated at 37°C for 18 h. Up to six isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC on the respective media, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, mRBA had the highest sensitivity (99.2%), correctly detecting a significantly higher proportion of STEC serogroups than either CS (79.4%; P < 0.05) or mPossé2 (91.7%; P < 0.05). mRBA also had the highest negative predictive value (90.0%), correctly identifying a significantly higher proportion of true-negative samples compared with CS (25.7%; P < 0.05) and mPossé2 (46.2%; P < 0.05). However, mRBA also had the lowest analytical specificity of 83.2% (P < 0.05), yielding the lowest proportion of colonies tested that were STEC positive (3,548 of 4,263) compared with 97.7% (3,607 of 3,693) for mPossé2 and 98.0% (2,875 of 2,935) for CS. Reduced specificity results in more work and higher expense due to the increased number of colonies that must be tested. Further improvements in agar culture media for non-O157 STEC isolation are needed.

Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains producing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of pig postweaning diarrhea (PWD). We recently identified neutralizing epitopes of fimbriae K88 and F18, heat-labile toxin (LT), heat-stable toxins type I (STa) and type II (STb), and Shiga toxin 2e (Stx2e). In this study, we explored a novel epitope- and structure-based vaccinology platform, multiepitope fusion antigen (MEFA), for PWD vaccine development. By using an epitope substitution LT toxoid, which lacks enterotoxicity but retains immunogenicity, as the backbone to present neutralizing epitopes of two ETEC fimbriae and four toxins, we generated PWD fimbria-toxin MEFA to mimic epitope native antigenicity. We then examined MEFA protein immunogenicity and evaluated MEFA application in PWD vaccine development. Mice subcutaneously immunized with PWD MEFA protein developed strong IgG responses to K88, F18, LT, and STb and moderate responses to the toxins Stx2e and STa. Importantly, MEFA-induced antibodies inhibited adherence of K88 or F18 fimbrial bacteria to pig intestinal cells and also neutralized LT, STa, STb, and Stx2e toxicity. These results indicated that PWD fimbria-toxin MEFA induced neutralizing antibodies against an unprecedent two fimbriae and four toxins and strongly suggested a potential application of this MEFA protein in developing a broadly protective PWD vaccine.IMPORTANCE ETEC-associated postweaning diarrhea (PWD) causes significant economic losses to swine producers worldwide. Currently, there is no effective prevention against PWD. A vaccine that blocks ETEC fimbriae (K88 and F18) from attaching to host receptors and prevents enterotoxins from stimulating water hypersecretion in pig small intestinal epithelial cells can effectively protect against PWD and significantly improves pig health and well-being. The fimbria-toxin MEFA generated from this study induced neutralizing antibodies against both ETEC fimbriae and all four ETEC toxins, suggesting a great potential of this fimbria-toxin MEFA in PWD vaccine development and further supporting the general application of this novel MEFA vaccinology platform for multivalent vaccine development.

Intimate Attachment of Escherichia coli O157:H7 to Urinary Bladder Epithelium in the Gnotobiotic Piglet Model.

Enterohemorrhagic Escherichia coli (EHEC), a pathogenic subset of Shiga toxin-producing E. coli (STEC), is an important cause of hemorrhagic colitis and hemolytic-uremic syndrome (HUS), and a rare cause of urinary tract infections (UTIs) with associated HUS. EHEC strains attach intimately to intestinal epithelium with formation of actin pedestals (attaching-effacing (A/E) lesions); however, the mechanism of EHEC attachment to the uroepithelium is unknown. We conducted a retrospective study on archived urinary bladder specimens from gnotobiotic piglets that naturally developed cystitis associated with EHEC O157:H7 infection following oral inoculation and fecal shedding. Paraffin-embedded bladder tissues from three piglets with cystitis and immunohistochemical evidence of EHEC O157:H7 adherence to the uroepithelium were processed for and examined by transmission electron microscopy. EHEC O157:H7 bacteria were found in one of three piglets, intimately attached to pedestals on the apical surfaces of the superficial urothelium (umbrella cells). Cystitis was significantly associated with the length of survival of the piglets post-inoculation (p = 0.0339; estimated odds ratio = 2.6652). This is the first report of E. coli causing A/E-like lesions in the uroepithelium, and also evidence of the utility of the gnotobiotic piglet as a model for studies of the pathogenesis of EHEC UTIs.

Investigation of congestive heart failure in beef cattle in a feedyard at a moderate altitude in western Nebraska.

Right-sided congestive heart failure (brisket disease) commonly occurs in cattle raised at elevations >2,500-3,500 m. We investigated clinical cases resembling brisket disease at a western Nebraska feedyard at a moderate altitude (1,369 m). Over a 15-mo period (2009-2010), we examined 17 cases (16 steers and 1 heifer), all purebred Angus. All animals had clinical right-sided heart failure: brisket and ventral abdominal edema, and severe chronic passive congestion of the liver. Gross examination confirmed right ventricular hypertrophy (left ventricle plus septum: right ventricle weight ratio mean: 1.33 vs. 2.8-4.0 reference interval). Microscopically, all 17 cases had interstitial fibrosis (mean score: 2.4 ± 0.8) and 6 had replacement fibrosis of the right ventricle, whereas 14 had interstitial fibrosis (mean score: 1.2 ± 0.2) and 0 had replacement fibrosis of the left ventricle. Lesions of arteriosclerosis were seen in 9 of 16 cases in 51 of 571 (8.9%) right ventricular coronary arteries, and in 10 of 16 cases in 52 of 366 (14.2%) left ventricular coronary arteries. The probability of coronary arteriosclerosis was greater in papillary ventricular muscle (OR = 11.3; p < 0.0001), left ventricle (OR = 4.8; p < 0.0001), and larger arteries (OR = 1.01; p < 0.0001). Pulmonary arteries and arterioles had lesions compatible with hypoxia-induced pulmonary hypertension. We hypothesize that moderate hypobaric conditions significantly contributed to disease in cattle genetically predisposed to hypoxia-induced pulmonary hypertension. Adiposity, coronary arteriosclerosis, and left ventricular fibrosis may have contributed to the condition; however, the cattle died prior to development of advanced obesity.

Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG.

Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC).

K88 and F18 fimbrial enterotoxigenic Escherichia coli (ETEC) are the major causes of post-weaning diarrhea (PWD) in pigs. A vaccine that induces broad immunity to prevent K88 and F18 fimbrial ETEC bacterial attachment and colonization in pig small intestines and to neutralize enterotoxin enterotoxicity would be effective for PWD. Structure-based multiepitope-fusion-antigen (MEFA) technology using a backbone immunogen to present neutralizing epitopes of representing virulence factors capacitates development of broadly protective ETEC vaccines. Neutralizing epitopes have been identified from K88 fimbrial adhesin (FaeG) and enterotoxins but not F18 fimbrial adhesin. In this study, we in silico identified immunodominant epitopes from F18ac fimbrial subunit FedF which plays a critical role in F18 fimbrial adherence, genetically fused each epitope to a carrier, examined immunogenicity of each epitope fusion, and determined epitope-derived antibodies neutralizing activities against F18 fimbrial adherence. Data showed that seven immune-dominant epitopes were identified from FedF subunit. Fused to heterologous human ETEC adhesin subunit CfaB, epitope fusions induced anti-F18 antibodies in subcutaneously immunized mice. Moreover, antibodies derived from each fusion significantly blocked adherence of a F18-fimbrial E. coli bacteria to pig intestinal cell line IPEC-J2. While all seven epitopes exhibited neutralizing activity, results from this study identified FedF epitopes #3 (IPSSSGTLTCQAGT) and #7 (QPDATGSWYD) the most effective for antibodies against F18 fimbrial adherence, and suggested their future application in PWD vaccine development.

Tellurite Resistance in Shiga Toxin-Producing Escherichia coli.

Potassium tellurite (K2TeO3) is an effective selective agent for O157:H7 Shiga toxin-producing Escherichia coli (STEC), whereas tellurite resistance in non-O157 STEC is variable with information on O45 minimal. High-level K2TeO3 resistance in STEC is attributable to the ter gene cluster with terD an indicator of the cluster's presence. Polymerase chain reactions for terD and K2TeO3 minimum inhibitory concentration (MIC) determinations in broth cultures were conducted on 70 STEC and 40 non-STEC control organisms. Sixty-six STEC strains (94.3%) were terD+ compared to 28 control organisms (70.0%; P < 0.001). The prevalence of terD in O103 STEC strains was 70%, whereas in all other serogroups it was ≥ 90%. The K2TeO3 geometric mean MIC ranking for STEC serogroups from highest to lowest was O111 > O26 > O145 > O157 > O103 > O121 = O45. The K2TeO3 geometric mean MIC was significantly higher in terD+ than in terD- STEC, but not in terD+ versus terD- control strains. Resistance to K2TeO3 (MIC ≥ 25 mg/L) was exhibited by 65/66 terD+ and 0/4 terD- STEC strains, compared to 12/28 terD+ and 8/12 terD- control strains. These results confirm previous studies showing the significantly higher prevalence of the ter gene cluster in STEC strains, and the relationship between these genes and K2TeO3 resistance in STEC and especially intimin (eae)-positive STEC, in contrast to non-STEC organisms. O45 and O121 STEC, although frequently terD positive, on average had significantly lower levels of K2TeO3 resistance than O26, O111, and O145 STEC.

Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden.

Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.

Culture-Based Quantification with Molecular Characterization of Non-O157 and O157 Enterohemorrhagic Escherichia coli Isolates from Rectoanal Mucosal Swabs of Feedlot Cattle.

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens carried in the intestinal tracts of ruminants and shed in the feces. High concentrations (≥104 colony-forming units [CFU]/g) of EHEC in cattle feces are associated with contamination of hides, and subsequently, carcasses and beef. Several studies using agar media have quantified O157 but few have quantified non-O157 EHEC in samples from cattle. Thus, the objective of this study was to determine the concentration of O157 and non-O157 EHEC in cattle, and to characterize the associated EHEC isolates for their virulence potential. Two hundred feedlot steers were sampled by rectoanal mucosal swab (RAMS) every 35 days over four sampling periods, and a spiral plating method using modified Possé differential agar was used to quantify EHEC organisms in these samples. Bacterial colonies from agar plates were tested by multiplex PCR for Shiga toxin and intimin genes (stx and eae, respectively), and confirmed EHEC isolates (i.e., positive for both stx and eae) were serotyped and characterized for virulence genes using a microarray. Organisms detected in this study included O26, O101, O103, O109, O121, O145, O157, and O177 EHEC, with all except O121 quantifiable and measuring within a range from 9.0 × 102 to 3.0 × 105 CFU/g of RAMS sample. Organisms of the same EHEC serogroup were not detected in quantifiable concentrations from a single animal more than once. EHEC organisms most commonly detected at quantifiable levels were O26, O157, and O177. Interestingly, O26 EHEC isolates tested negative for stx1 but positive for stx2a. High concentrations of EHEC were detected in 11 (5.5%) of the steers at least once over the sampling period. These results indicate that in addition to O157, non-O157 EHEC are transiently present in high concentrations in the rectoanal mucosal region of cattle.

Efficacy of Urtoxazumab (TMA-15 Humanized Monoclonal Antibody Specific for Shiga Toxin 2) Against Post-Diarrheal Neurological Sequelae Caused by Escherichia coli O157:H7 Infection in the Neonatal Gnotobiotic Piglet Model.

Enterohemorrhagic Escherichia coli (EHEC) is the most common cause of hemorrhagic colitis and hemolytic uremic syndrome in human patients, with brain damage and dysfunction the main cause of acute death. We evaluated the efficacy of urtoxazumab (TMA-15, Teijin Pharma Limited), a humanized monoclonal antibody against Shiga toxin (Stx) 2 for the prevention of brain damage, dysfunction, and death in a piglet EHEC infection model. Forty-five neonatal gnotobiotic piglets were inoculated orally with 3 × 108 colony-forming units of EHEC O157:H7 strain EDL933 (Stx1⁺, Stx2⁺) when 22-24 h old. At 24 h post-inoculation, piglets were intraperitoneally administered placebo or TMA-15 (0.3, 1.0 or 3.0 mg/kg body weight). Compared to placebo (n = 10), TMA-15 (n = 35) yielded a significantly greater probability of survival, length of survival, and weight gain (p <0.05). The efficacy of TMA-15 against brain lesions and death was 62.9% (p = 0.0004) and 71.4% (p = 0.0004), respectively. These results suggest that TMA-15 may potentially prevent or reduce vascular necrosis and infarction of the brain attributable to Stx2 in human patients acutely infected with EHEC. However, we do not infer that TMA-15 treatment will completely protect human patients infected with EHEC O157:H7 strains that produce both Stx1 and Stx2.

Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli.

Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx 1 , stx 2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.